Some Recent Advances in Hepatobiliary Disease and in the Clinical Aspects of Bile Acids Held at Bristol Royal Infirmary , 22 September 1989 FALLING INTO

are misleading and likely to lead to faulty ideas in attributing both etiologic causes to the stones, and potential for succesful dissolution. It is now generally agreed that all stones in gallbladders harboring multiple stones are of approximately the same composition. Further, if one plots percent of patients undergoing elective cholecystectomy for biliary pain, but without complications (i.e. no infection) versus per cent cholesterol in a single stone obtained from each gallbladder, one obtains a bimodal frequency distribution nomogram. In Western countries approximately 25% of gallstone patients have stones with <30% cholesterol by weight?most with <10% cholesterol. These are pigment gallstones and are composed of amorphous calcium bilirubinates that have undergone polymerization and oxidation, calcium carbonate and less frequenly calcium phosphate. Provided the bile is sterile and the stones contain no evidence of previous bacterial infection of bile (e.g. absence of bacterial rhamnose), no

The time honored classification of gallstones, attributed to Morgagni (1682-1771), Professor of Anatomy in Padua, labels gallstones according to what appears to be their predominant constituent, that is cholesterol or bile pigment, or a mixture of these substances. In this connection appearances are misleading and likely to lead to faulty ideas in attributing both etiologic causes to the stones, and potential for succesful dissolution. It is now generally agreed that all stones in gallbladders harboring multiple stones are of approximately the same composition. Further, if one plots percent of patients undergoing elective cholecystectomy for biliary pain, but without complications (i.e. no infection) versus per cent cholesterol in a single stone obtained from each gallbladder, one obtains a bimodal frequency distribution nomogram. In Western countries approximately 25% of gallstone patients have stones with <30% cholesterol by weight?most with <10% cholesterol. These are pigment gallstones and are composed of amorphous calcium bilirubinates that have undergone polymerization and oxidation, calcium carbonate and less frequenly calcium phosphate. Provided the bile is sterile and the stones contain no evidence of previous bacterial infection of bile (e.g. absence of bacterial rhamnose), no stones contain a percentage of cholesterol between 30 and 50% by weight. The remainder of the gallstone patients ~75%, possess stones with more than 50% cholesterol by weight. Further, the percent of patients in each decile, increases stepwise reaching a maximum for stones containing 90-100% cholesterol. Because anerobic bacterial infection of bile leads to calcium soap, calcium bile salt, calcium bilirubinate and even cholesterol precipitation, stones obtained from infectious bile, will have a continuum in percent cholesterol and hence are not classifiable on the basis of the aforegoing criteria. These stones are most often primary ductal in origin and are 'trivially' classified as 'brown' pigment stones. In contrast, the pigment stones formed in sterile gallbladder bile are 'black'. While brown 'stones' invariably contain precipitated calcium bilirubinate, the major components of 'brown' stones may be mucin glycoproteins, dead bacteria, calcium soaps, other calcium salts, deconjugated bile acids etc. These infectious stones will not be further discussed here, rather my focus will be upon the metabolic and physical-chemical origins of the two categories of gallbladder stones.

PHYSICAL CHEMISTRY
Common to all gallbladder stones either cholesterol or pigment is supersaturation, an essential but not sufficient physical-chemical defect. Because cholesterol (Ch) and monoanionic unconjugated bilirubinate (HUCB ) are only sparingly soluble in water, they are transported in bile by binding to the major biliary lipids which are bile salts and diacylphosphatidylcholines (lecithins). Bile salt are present as monomers (equivalent to the critical micellar concentration), simple micelles (polymolecular aggregates without lecithin) and mixed micelles (polymolecular aggregates with lecithin); whereas lecithin which is otherwise insoluble in water, is solubilized as mixed micelles (with bile salts) and dispersed as unilamellar vesicles (closed bilayered membrane structures) containing little or no bile salts. Bile salt monomers, as well as the lipid aggregates can bind both Ch and HUCB~ and act as their 'carriers' in bile. Hence supersaturation can occur if there is an excess of biliary Ch or HUCBor if there is a deficiency in the 'carriers'. From secretory studies, we now know that all Ch gallstone patients have either hypersecretion of hepatic Ch or hyposecretion of bile salts into bile. Rarely a combination of both secretory defects may occur. Once bile salt monomer plus simple and mixed micellar solubility of Ch is exceeded, lecithin vesicles appear in bile and disperse, albeit in an unstable way, the excess Ch molecules as lecithin/Ch vesicles. If these become unstable as they do in Ch gallstone patients they aggregate and fuse, grow in size and after a couple of days they 'spawn' Ch monohydrate crystals. Finally the high Ch content of vesicles leads to continued growth of the microcrystals to produce crystals of sufficient size to agglomerate into macroscopic gallstones.
While a key physical chemical characteristic of Ch stone biles is the presence of vesicles carrying excess Ch, pigment stone biles, in contrast, are uniformly devoid of lecithin/Ch vesicles. This immediately suggests that pigment stone gallbladder biles are missing an important carrier not only for binding HUCB but also for binding ionic calcium (Ca:+).
The absence of these carriers may be due to hypersecretion of bile salts (which appears unlikely) or hyposecretion of hepatic Ch. While secretory and production studies of HUCB" in human biles are lacking, there appears to be an absolute increase in biliary UCB (which forms HUCB at peri-neutral biliary pH values), either from hypersecretion or de novo formation from bilirubinate conjugates, the primary form in which bilirubin is secreted into bile. Since vesicles are absent in such biles, the nucleation sequence described for Ch, cannot take place but rather, precipitation of Ca(HUCB )-, must occur from HUCB? supersaturated bile salt monomers and from simple and mixed micelles.

PATHOGENESIS
Formation of either Ch or pigment stones in the gallbladder are not single diseases but the complications of many different diseases that have stones as their common physicalchemical endpoints.

CHOLESTEROL GALLSTONES
A. Multiple etiologic factors in hypersecretism of biliary Ch are (i) increased Ch input to the liver from increased hepatic uptake of low density lipoproteins (LDL), high density lipoproteins (HDL) and chylomicron remnants such as in estrogen-and dietary-induced gallstones. (ii) Increased Ch synthesis in the liver (and extrahepatic tissues) as in gallstones secondary to human obesity and hypertriglyceridemic states. (iii) Decreased Ch catabolism in the liver either to form bile salts de novo or cholesteryl esters for storage, nascent HDL and very low density lipoprotein (VLDL) production (as in progestogenic and 'fibric acid1 induced gallstones). Multiple etiologic factors in hyposecretion of bile salts are (i) decreased de novo bile salt synthesis such as in Ch gallstones caused by human aging, and congenital and acquired bile salt synthetic defects. (ii) decreased enterohepatic cycling of bile salts from motor, hormonal or receptor defects ("idiopathic"; these defects have not been defined at the molecular level). (iii) Torrential gastrointestinal loss of bile salts uncompensated by de novo hepatic synthesis. Although this disorder has been claimed as responsible for Ch gallstone formation in chronic ileal disease (Crohn's), ileal resection, bypass, or acidic small bowel states (Cystic Fibrosis), it now appears that pigment gallstones are the final endresult of these diseases. (i) Any chronic hypercalcemic states, (e.g. hyperparathyroidism, sarcoidosis, immobilization etc.).
(ii) Diminished binding to/or absent vesicles, especially from hyposecretion of biliary Ch (e.g. alcoholic cirrhosis) (iii) Decreased binding to micelles from hyposecretion of bile salts.

MODIFIERS OF NUCLEATION
Modifiers are kinetic and motor defects that are essential for the thermodynamic defect of supersaturation to result in gallstones. These defects are best defined for Ch crystal nucleation and gallstone formation. Present in normal bile are anti-nucleating glycoproteins as well as possibly other substances, some of which prevent vesicle fusion and aggregation and nucleation, whereas others appear to be anti-crystal growth substances. It is currently thought that these potent inhibitors are absorbed to vesicles or to the active growth sites on the Ch-crystal surfaces where they block further crystal growth and perhaps suppress aggregation. In Ch stone biles, there appears to be both excess of mucin glycoproteins as well as soluble glycoproteins that act as powerful accelerators of vesicle fusion, growth and crystal nucleation. Initially Ch crystals, within the gallbladder, are minute and possibly too small to be trapped in the viscus, unless other modifiers are present concurrently. This appears to be the case. In Ch gallstone patients there is primary gallbladder hypomotility of uncertain etiology. Hypomotility may be a primary or secondary smooth muscle defect, occurring in response to some intravesicle chemical "trigger" that is absorbed by the mucosa. In some patients hypomotility may be related to defective cholecystokinin release from the upper small intestine or the gallbladder's response to cholecystokinin. The result is a decreased and delayed gallbladder ejection fraction and increased residual volume.
A further list of factors promoting retention of minute Ch crystals and subsequent gallstone formation are (i) Ch monohydrate is more dense than bile; the Ch crystals sink to the fundus of the gallbladder and cannot reach the cystic duct since this outlet is at the top of the viscus when the subject is upright, (ii) Mucin gel presumably aids in the retention of crystals at the gallbladder's fundus since mucin also depends upon normal gallbladder contraction for its elimination (iii) Mucin gel may also act as intercrystalline cement facilitating agglomeration of small Ch microcrystals. (iv) The site of nucleation is most likely not in the fluid intravesicle 'phase' of bile but juxtamucosal in the visco-elastic mucin gel on the gallbladder wall. It is here that mucin gel is first formed and where Ch-supersaturated vesicles and micelles of bile are concentrated by solvent drag secondary to dehydration of bile. Hence the organic mucin matrix, gallbladder hypomotility, as well as other uncharacterized substances my greatly modify and facilitate the chances of Ch crystal formation and retention within the gallbladder.Whether kinetic and motor defects modify calcium bilirubinate formation and precipitation within the gallbladder are unknown but they remain a real possibility. Clearly, the more detailed our knowledge of all the factors involved in the progression of abnormal bile to the stone 'end-point' the more developed our understanding of the pathogenesis of stones will become. This is particularly important for developing effective programs for the prevention of both cholesterol and pigment stone formation in human beings. All subjects were questioned about their habitual intake of sugar-and fibre-containing foods and fasting plasma was analysed for insulin, glucose, total cholesterol^ triglycerides, HDL2 and HDL, cholesterol. Information was also obtained on parity (including abortions), weight change since age 20, waist and hip circumferences, bowel habit, and usual stool form on a validated scale from 1 (scybala) to 7 (watery). In a parallel case-control study, 80 subjects with unsuspected gallstones are being compared with age-and sexmatched stone-free controls with respect to whole-gut transit time and nutrient intakes (assessed from a 4-day weighed record).
Analysis of these extensive data is proceeding. By mid-September 1989 the following facts had emerged: (1) Gallstone prevalence flattens off in middle-aged women in Bristol; otherwise prevalence rises with age in both sexes, reaching 22.4% in women aged 60-69; (2) the incidence of biliary pain was very low in people with gallstones and even lower (<5%) if the standardised questionnaire was supplemented by normal historytaking; (3) women are more likely than men to have symptoms from their gallstones and to undergo cholecystectomy.

THE BILIARY TRACT IN PATIENTS WITH GALLSTONE PANCREATITIS
C. P. Armstrong, Dept of Surgery, Bristol Royal Infirmary The concept of gallstone migration is now generally accepted although the reason why only a few patients with gallstones develop pancreatitis is unknown. One thousand five hundred patients undergoing surgery for gallstones were prospectively studied; 203 (13.5%) had a history of gallstone pancreatitis. Patients with pancreatitis had significantly more gall bladder stones 22.3? 14.1 versus 15.2? 11.6, P<0.02, and 74% had more than 10 stones in contrast to 43% control patients (P<0.001). The smallest gall bladder stone was considerably smaller in the pancreatitis patients; 2.4?1.6 mm versus 5.9?4.1, P<0.001; and 93% had stones of 3 mm or less contrary to 37% of controls (P<0.001). The cystic duct was wider in pancreatitis patients; 4.5?2.0 mm versus 3.3?1.9mm, P<0.001; and 58% had a cystic duct of 4 mm or larger compared with 17 per cent of controls (P<0.001). Stone passage through the cystic duct was possible in all patients with pancreatitis but in only half the controls. The common bile duct diameter in patients with pancreatitis was independent of stones within its lumen. Choledochal calculi were of equal size in the two groups. Pancreatic-duct reflux was far more commonly observed on the operative cholangiograms of pancreatitis patients; 67.0 versus 16.6%, PcO.OOOl.
These observations clearly affirm the importance of small stone migration in the pathogenesis of acute pancreatitis. Patients with gallstones who develop pancreatitis have a biliary tract that predisposes to gallstone migration and pancreatic-duct reflux. Ursodeoxycholic acid is normally a minor component of human bile. Treatment with ursodeoxycholic acid leads to enrichment of bile so that up to 50% of the bile acids become ursodeoxycholic acid. This reduces biliary cholesterol levels, and cholesterol gallstone formation can be prevented in risk populations. Existing gallstones can be dissolved. This is only very effective for small stones, so the additional use of extracorporeal shock wave lithotripsy to reduce the size of larger stones is an attractive option. The combined use of chenodeoxycholic acid with ursodeoxycholic acid may be even more effective therapy for gallstones, leading to more rapid and complete dissolution, and possibly reducing the risk of induced gallstone calcification which is the only definite sideeffect of ursodeoxycholic acid therapy. It was found by chance that gallstone patients with coexisting liver disease derived additional benefit from bile acid therapy. This is best established in primary biliary cirrhosis. Treatment with ursodeoxycholic acid 750 mg. daily improves symptoms like pruritus and diarrhoea. Abnormally elevated serum enzyme levels are reduced after a few weeks, and this fall continues until four to six months, the effects being sustained thereafter. No improvement in steatorrhoea was shown by triolein breath tests performed before and after four months' therapy, and the possibility of a deterioration could not be excluded. Liver histology did not usually change after four months, but there was sometimes a reduction in inflammatory cell infiltrate. The effect on survival is not known, and it will take many years to assess. There is no definite advantage in using larger doses of ursodeoxycholic acid than 750 mg. daily, but smaller doses may be just as effective. Though evidence is patchy and incomplete, benefit from ursodeoxycholic acid has also been reported in chronic active hepatitis, primary sclerosing cholangitis and biliary atresia, as well as in bile reflux gastritis and non-specific dyspepsia.
The only cure for primary biliary cirrhosis is liver transplantation. Colchicine and azathioprine may prolong life but results are equivocal. Attempts to control liver inflammation have usually involved toxic drugs like prednisolone, penicillamine, cyclosporin, methotrexate and nalmefene. Since ursodeoxycholic acid controls symptoms and appears to be harmless, it has a claim to be the treatment of first choice in primary biliary cirrhosis if any drug therapy is required. University Department of Surgery, Bristol Royal Infirmary Reflux oesophagitis is attributed to the digestive action of acid and pepsin on the oesophageal mucosa. Bile acids are also thought to play a role (and may be responsible for some treatment failures), but have not been studied in the oesophagus.
Fifty-two patients with gastro-oesophageal reflux, none of whom had previous gastric surgery, were studied by simultaneous oesophageal aspiration and pH monitoring between 1700-0900 hr. Aspirates, in 2 hourly aliquots, were assayed for conjugated and unconjugated bile acids and pepsin. The results were compared according to the degree of oesophagitis determined by endoscopy. Patients with oesophagitis had greater acid reflux than refluxers with a normal oesophagus (18 vs 8.3 for % time pH<4; P<0.05). Conjugated bile acids were found in aspirates from 75% of patients, and were equally distributed among the different grades of oesophagitis. Only 2% of 364 aspirates contained concentrations known to be cytopathic to oesophageal mucosa (>1 mmol/1), and no bile acids were unconjugated. Patients with complicated oesophagitis (stricture, Barrett's oesophagus) had higher pepsin concentrations than those with uncomplicated erosive oesophagitis (153 vs 46ug/ml P<0.001).
The results affirm the importance of acid and pepsin in the pathogenesis of reflux oesophagitis, and suggest that bile acids do not play a major role in this disease.  (1). In a population study faecal bile acid (FBA) concentration was shown to correlate positively with the incidence of CRC and later studies (2,3) showed a similar correlation in CRC case versus control studies. In brief, a hypothesis was presented and tested during the following decade based on the following dictat: "CRC may be caused by the production of carcinogenic compounds (unsaturated bile acids) from the metabolism of bile acids by intestinal bacteria within the large bowel". Although bacteria (especially Clostridia) were isolated and identified from intestinal contents capable of elaborating the necessary enzymes, the products, after exhaustive testing, were not found to be potent mutagens or carcinogens (4). However, in many "cancer" models it has been shown that the secondary bile acid metabolites principally lithocholic acid (LCA) and deoxycholic acid (DCA) exhibit both co-mutagenic and co-carcinogenic properties. It has been concluded, therefore, that the positive correlations observed in population studies between FBA concentration and CRC incidence is probably due to the cancer promoting properties of these lipids.

LARGE
With the advent of more sophisticated methods [Owen et al., 1984(5)] for lipid analyses, recent studies have shown that in CRC case control studies the faecal bile acid profile is more important than FBA concentration especially as a marker of CRC (6).
In summary, the LCA:DCA ratio in CRC patients (1.81 ?0.19) is diametrically opposed to that of control subjects (0.87?0.07) such that 72% of CRC patients have an abnormal ratio (>1:0) compared to only 25% of controls. Of interest is that the LCA:DCA ratio correlates positively with adenoma size and severity of CRC.
It is concluded that the LCA:DCA ratio may be a useful adjunct to future screening procedures for CRC.